The present invention relates to a method for measuring the amount of an objective constituent for measurement contained in a specific lipoprotein in a biological sample such as serum and plasma. More particularly, the invention provides a method for measuring the amount of cholesterol contained in high density lipoprotein (hereinafter referred to as HDL). The method of the present invention can be applied advantageously to automatically quantitative measurement of HDL cholesterol which has widely been used in the field of clinical tests.
As to methods for measuring the amount of the objective constituent for measurement contained in the specific lipoprotein such as cholesterol contained in HDL, there have been known various methods including ultracentrifugal separation method, electrophoresis method, precipitation method and the like. In the field of clinical tests, the precipitation method has usually and widely been carried out, for the reason that this method can be carried out simply as compared with the ultracentrifugal separation method and the electrophoresis method.
However, a problem with conducting the precipitation method is that this method cannot be carried out only by use of a conventional automatic analyzer, for the reason that this method comprises two steps; (1) a step of pretreatment operation, which comprises, a) mixing a serum with a precipitating agent and forming precipitates of the lipoproteins other than the specific lipoprotein, b) separating the precipitates by centrifugation, and c) collecting thus formed supernatant; and (2) a step of measuring the amount of the objective constituent contained in the specific lipoprotein in the supernatant.
In order to solve the problem, there have been developed some methods; for example Japanese Patent Kokai (Laid-open) No. Hei 6-242110 (1994) discloses a method in that, lipoproteins other than the specific lipoprotein are agglutinated by use of a precipitating agent and/or an antibody reactive to the lipoproteins other than the specific lipoprotein, then the specific constituent in the specific lipoprotein is subjected to reaction with a reagent for quantitative measurement thereof, and thereafter, at the same time, or after the reaction is stopped, the agglutinated lipoprotein is dissolved to give a homogeneous solution, and then the optical absorbance of said solution is measured.
This method, however, requires 3 or 4 kinds of reagents and thus can be applicable only to such remarkably limited automatic analyzers wherein measurement can be conducted with the use of 3 or 4 kinds of reagents, while this method cannot be applied to such automatic analyzers wherein only up to 2 kinds of reagents can be used as conventionally used in clinical tests. Further, this method has the defect of lower reproducibility of measurement because of using 3 or 4 kinds of reagents.
In consideration of the above circumstances, the problem to be solved by the present invention is to provide a method capable of measuring directly the amount of the objective constituents for measurement contained in the specific lipoprotein in a biological sample by using an automatic analyzer, without subjected to any pretreatment operation to separate the specific lipoprotein from other lipoproteins, which has necessarily been carried out in conducting the precipitation method widely used in conventional clinical tests.